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Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease
Biochemical and Biophysical Research Communications xxx (2017) 1e8
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Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease Mari Kim b, 1, Manivannan Subramanian a, 1, Yun-Ho Cho a, Gye-Hyeong Kim a, Eunil Lee b, Joong-Jean Park a, * a b
Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2017 Accepted 4 December 2017 Available online xxx
The accumulation and aggregation of phosphorylated tau proteins in the brain are the hallmarks for the onset of Alzheimer's disease (AD). In addition, disruptions in circadian rhythms (CRs) with altered sleepwake cycles, dysregulation of locomotion, and increased memory defects have been reported in patients with AD. Drosophila flies that have an overexpression of human tau protein in neurons exhibit most of the symptoms of human patients with AD, including locomotion defects and neurodegeneration. Using the fly model for tauopathy/AD, we investigated the effects of an exposure to dim light at night on AD symptoms. We used a light intensity of 10 lux, which is considered the lower limit of light pollution in many countries. After the tauopathy flies were exposed to the dim light at night for 3 days, the flies showed disrupted CRs, altered sleep-wake cycles due to increased pTau proteins and neurodegeneration, in the brains of the AD flies. The results indicate that the nighttime exposure of tauopathy/AD model Drosophila flies to dim light disrupted CR and sleep-wake behavior and promoted neurodegeneration. © 2017 Elsevier Inc. All rights reserved.
Keywords: Alzheimer's disease (AD) Circadian rhythm (CR) Sleep Dim light at night (dLAN) Neurodegeneration Drosophila melanogaster
1. Introduction Alzheimer's disease (AD) is one of the most common forms of dementia all over the world. The onset of AD is associated with the accumulation of b-amyloid peptide (Ab) and/or phosphorylated tau [1]. Tau stabilizes microtubules, which are involved in neuronal axon transport [2]. In patients with AD, hyperphosphorylated tau is segregated from microtubules, which results in the loss of microtubule integrity. In addition, hyperphosphorylated tau accumulates in patients with AD and forms intracellular neurofibrillary tangles (NFTs), which become neurotoxic and eventually cause neurodegeneration [3]. Rhythmic behaviors are important in normal physiological processes. Two common and interacting rhythmic behaviors are circadian rhythms (CRs) and sleep-wake behaviors. CRs are involved in the regulation of the sleep-wake behavior, and disruptions in CRs eventually result in changes in sleep. Normal sleep
* Corresponding author. E-mail address: [email protected] (J.-J. Park). 1 Contributed equally on this work.
is required to restore neural plasticity and function [4] and clear toxic compounds that accumulate during the daytime [5]. Studies conducted using AD model mice have shown that the CRs of the mice are altered by the accumulation of Ab and the formation of NFTs [6]. Sleep is profoundly affected in patients with AD showing altered sleep-wake behavior with reduced night sleep. Mouse models exhibiting AD phenotypes also show decreased sleep at night [6]. Thus, sleep disturbances are considered an indicator of the pathogenesis of AD [7]. Even in healthy individuals, decreased sleep enhances the accumulation of Ab peptides and the formation of NFTs [8]. Thus, sleep and AD are interlinked, and any perturbations of one will affect the other [9]. The interference of CRs and/or sleep by light at night is referred to as light pollution, which has recently emerged as a major health problem in many developed countries, where any nighttime exposure to light that is over 10 lux in a residential area is prohibited. In modern industrialized societies, shift workers are continuously exposed to light at night, which disrupts their CRs and sleep-wake behaviors [10]. Shift workers have recently been reported to be more susceptible to cardiovascular disease, diabetes, obesity, cancer, and metabolic disorders [11,12]. Studies conducted
https://doi.org/10.1016/j.bbrc.2017.12.021 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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M. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
on mice have also shown that disruptions of CRs by exposure to dim light (10 lux) at night leads to increased tumorigenesis with reduced lifespans [11]. Because CRs are regulated by the nervous system, any negative effects of CR disruptions on other systems should appear rapidly. Drosophila melanogaster has been serving as a useful model system for investigating the genetic bases of CRs and sleep [13] and the pathogenesis of AD [14,15]. Previous studies on Drosophila have reported that exposure to moonlight at night alters CRs [16]. However, it remains unclear whether exposure to 10 lux of dLAN affects rhythmic behaviors and/or neurodegeneration in AD model organisms. In this study, we investigated the effects of short-term exposures to dim light on physiology in AD flies and found that AD flies were more vulnerable to the negative effects of the dim light exposure.
used as a loading control. 2.5. Immunohistochemistry Adult fly heads were fixed in 4% paraformaldehyde and washed with PBS. The brains were blocked with 5% normal goat serum in PBS containing 0.1% Triton-X 100 (PBST) and incubated with murine anti-tau and rabbit anti-pTau antibodies for 48 h at 4 C. After washing with PBST, the samples were incubated with an antimouse secondary antibody conjugated with Alexa Fluor 488 and an anti-rabbit secondary antibody conjugated with Alexa Fluor 524 overnight at 4 C. After washing, brains were mounted using Vectashield (Thermo Fisher Scientific Inc.) and images were acquired using confocal microscopy (Carl Zeiss Microscopy). 2.6. Brain histology
2. Materials and methods 2.1. Drosophila strains The flies were cultured on cornmeal-based standard food under a 12:12-h light-dark (LD) cycle at 25 C with 40e60% relative humidity [17]. Elav-GAL4 (ElavC155-GAL4) and GMR-GAL4 fly stocks were obtained from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Other stocks used were UAS-hTauWT, UAS-hTauRW [18], and w1118 (þ/þ) for the wild type. 2.2. Circadian rhythms and sleep Freshly eclosed Drosophila males were collected and transferred into 3-mm-diameter glass tubes containing food at one end. These flies were monitored for 6 days using the Drosophila Activity Monitoring System (TriKinetics, Inc., Waltham, MA, USA) in an incubator with a 12:12-h LD cycle. For the first 3 days, the flies were maintained under a normal LD cycle, and then exposed 10 lux during the dark cycle for the next 3 days. Locomotor activity was recorded for 30-min intervals, and rhythmicity and free-running periods were calculated using a cosinor analysis. Sleep behaviors were analyzed during the 3rd day of each lighting pattern (LD3 or LL3) using pySolo software. Sleep deprivation was calculated by subtracting the sleep time recorded on the third day (LD3 and LL3) during the 10-lux light exposure from the sleep time recorded during 0 lux in each treatment group. Similarly, the amount of sleep recovered from ZT09 to ZT21 the day after the third day was calculated [19].
The Drosophila heads that were fixed in 4% paraformaldehyde were embedded in paraffin blocks and sectioned at a thickness of 6 mm. These sections were mounted on slides and stained using hematoxylin and eosin. Quantification of the neurodegeneration was performed as mentioned by Lijima et. Al (2008) [14]. 2.7. Climbing assay Around 20 flies of each genotype were placed in a 50-mL glass mass cylinder and gently tapped to the bottom. The number of flies that climbed to the top was counted after 10 s. The data were acquired from three independent sets and then analyzed and plotted. 2.8. Data analysis The longevity data were used to generate Kaplan-Meier survival plots, and median lifespan was calculated. Statistical significance between the genotypes was analyzed using Log-Rank tests and the Prism software. For the data on the CR, sleep parameters, sleep deprivation and sleep recovery were analyzed using a 2-way analysis of variance with the main factors of genotype and light condition. The number of vacuoles and pTau intensity of the western blots were analyzed using a 2-way repeated analysis of variance. P values less than 0.05 were considered significant, and the data are presented as mean ± standard error of the mean. 3. Results 3.1. Accumulation of hTau negatively affected lifespan
2.3. Lifespan assay The flies were raised at 25 C under 50% relative humidity and a 12:12-h LD cycle, and the food vials were replaced every 2e3 days [17]. Dead flies were counted every 2e3 days, and the survival curves were plotted using Prism software (v6.05; GraphPad Software). 2.4. Western blotting Adult fly heads were homogenized in HEPES-EDTA lysis buffer and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and loaded onto SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk and incubated overnight at 4 C with anti-tau and antipTau antibodies (both 1:1000; Thermo Fisher Scientific) followed by incubation with secondary antibody (1:10,000; Cell Signaling Technology) and visualized with chemiluminescent detection. A mouse b-actin antibody (1:1000; Cell Signaling Technology) was
Studies of hTau have shown that the accumulation of hTau in fly brains shortens their lifespan and increases neurodegeneration in Drosophila [18]. Two types of human tau (hTau) protein were overexpressed: wild-type hTau (hTauWT) and mutant hTau, in which Arg406 was replaced by Trp (hTauRW). Accumulation of hTau proteins in the brain were confirmed with both immunohistochemistry (Fig. 1A) and western blotting (Fig. 1B). Flies overexpressing hTauWT exhibited the distinct rough eye phenotype under control of either the Elav-GAL4 or the GMR-GAL4 (Fig. 1C). The rough eye phenotype was exacerbated in hTauRW flies overexpressed both in neurons and the eye. Flies overexpressing hTauWT or hTauRW, in neurons showed significant shorter lifespans compared to controls (p < 0.001, Fig. 1D). The median lifespan (20 days) of the hTauRW-overexpressing flies was significantly shorter than that (39 days) of the hTauWT-overexpressing flies (p < 0.001, Fig. 1D). Furthermore, these results suggested that the neuronal overexpression of the hTau gene enhanced the formation of pTau and reduced lifespan in Drosophila.
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
M. Kim et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
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Fig. 1. Overexpression of hTau results in the accumulation of pTau and reduces lifespan. (A) Visualization of hTau in brains by immunohistochemistry. (B) Detection of hTau and pTau by western blotting. (C) Rough eye phenotypes in flies expressing hTauWT and hTauRW using GMR-GAL4 and Elav-GAL4. (D) The overexpression of either hTauWT or hTauRW in the brain using the pan-neuronal Elav-GAL4 (Elav/þ>hTauWT/þ; Elav/þ>hTauRW/þ) significantly reduced lifespan, compared to controls.
3.2. Negative effects of exposure to dim light at night on circadian rhythm in hTau-overexpressing flies Recent studies of AD models have investigated the disruptions in circadian behavior and locomotion in AD [20]. Circadian oscillations are controlled by both external and internal factors. To investigate whether internal rhythmicity was maintained in tauopathy flies, hTau-overexpressing flies were conditioned to a darkdark (DD) cycle, and their CRs were analyzed. Under the DD condition, the flies overexpressing hTauWT or hTauRW in neurons maintained their CRs, which were similar to those of the wild-type flies (F(2,54) ¼ 2.182, p ¼ 0.1227; Fig. 2A). Circadian rhythmicity was calculated using cosinor analyses, which measures period (time taken to complete one cycle in 24 h), amplitude (maximum activity in 24 h), and robustness (persistent behavior of locomotion). The cosinor analyses of their CRs showed that the period and amplitude of the locomotion behavior did not differ significantly between the LD and DD conditions in the hTau-overexpressing flies and the robustness under the LD condition was similar to that of the DD condition (Table 1). These results suggested that the intrinsic circadian clock mechanism was intact and present in both the hTauWT and hTauRW-overexpressing flies. Because the internal clocks functioned normally under the DD condition in the AD model flies, these flies were subjected to variations in external cues by exposing them to dLAN (10 lux). During exposure to 0 lux, normal behavior with the characteristic morning and evening peaks according to the lighting schedule was observed in the wild-type flies (Fig. 2B). However, the morning peak was reduced due to increased nocturnal locomotor activity on exposure to dLAN (Fig. 2B), whereas the CR period was unaffected (Table 2). Interestingly, under 10 lux, period was significantly decreased in the hTauWT-overexpressing flies (F(2,52) ¼ 7.672, p ¼ 0.0012; Table 2), whereas their robustness and amplitude were not significantly different (Table 2). In hTauRW-overexpressing flies, the dim light exposure resulted in a significant reduction in period (F(1,52) ¼ 9.022, p ¼ 0.0041; Table 2) and increased robustness
(F(2,52) ¼ 12.52, p < 0.0001; Table 2). Under dLAN, flies overexpressing hTauRW showed increased circadian disruption (Fig. 2B) with increased robustness. Despite these changes, climbing defects were observed only in hTau-overexpressing flies, and dim light at night did not affect their antigeotactic locomotor ability (Fig. 2C), which suggested that dim light at night only affected the circadian locomotor activity. 3.3. Disruption of sleep-wake behavior after exposure to dLAN in hTau-overexpressing flies Sleep, which is regulated by both circadian and homeostatic mechanisms, is disrupted in patients with AD. Sleep-wake behaviors were further assessed in hTau-overexpressing flies. Under the 0-lux condition, the wild-type flies exhibited normal sleep-wake behavior (Fig. 3A). However, they showed altered sleep-wake behaviors with decreased night-time sleep after exposure to dLAN (Fig. 3B) compared to flies under LD condition (F(5,45) ¼ 5.957, p ¼ 0.0003; Fig. 3E). However, they showed increased compensatory sleep during the daytime due to their sleep disturbances induced by the dLAN (F(5,45) ¼ 12.39, p < 0.0001; Fig. 3D). Flies overexpressing hTauWT showed no changes in sleep-wake behavior after exposure to dLAN (Fig. 3CeF). However, flies overexpressing hTauRW exhibited altered sleep-wake behavior (Fig. 3A) with significant reduction in total sleep (F(5,45) ¼ 9.536, p < 0.00019; Fig. 3C), night sleep (F(5,45) ¼ 5.937, p ¼ 0.0003; Fig. 3E), and sleep episodes at night (F(5,45) ¼ 2.736, p ¼ 0.0305; Fig. 3F) on exposure to dLAN. When the wild-type flies were exposed to the dLAN, their sleep was significantly deprived, but it was recovered the next day (Fig. 3G and H). However, the flies overexpressing hTauWT had significantly reduced sleep deprivation (p ¼ 0.0065; Fig. 3G) and recovery (p ¼ 0.013; Fig. 3H). For the hTauRW-overexpressing flies, considerable sleep deprivation was found, and the sleep amount that was recovered the next day was significantly reduced (p ¼ 0002; Fig. 3H). These data suggested that the exposure of AD flies to dim light affected their CR and sleep-wake behaviors.
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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Fig. 2. Disruption of CR in flies overexpressing hTau in the brain after exposure to dLAN. (A) CR in w1118 flies and hTau overexpressing flies under LD and DD conditions with cosinor analysis (Table 1). (B) CR in w1118 flies and hTau overexpressing flies under 0 lux and 10 lux conditions with cosinor analysis (Table 2). (C) Reduction of anti-geotactic locomotor (climbing) activity in hTau overexpressing flies under both 0 lux and 10 lux.
Table 1 Circadian rhythmicity under constant darkness condition. Light condition
LD
Genotype
w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
DD w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
Period Robustness Amplitude
25.40 1.53 11.00
24.20 3.72b 11.20
24.20 8.79b 16.08
22.4a 2.75 9.75
23.60 4.09 9.07
24.79b 16.18b,c 15.71
Significant differences compared between LD and DD conditions (a), compared with w1118 (b) and compared between Elav/þ>hTauWT/ þ and Elav/þ>hTauRW/ þ (c) (p < 0.05).
Table 2 Circadian rhythmicity on exposure to dim (10 lux) light at night. Light condition
0 lux
Genotype
w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
10 lux w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
Period Robustness Amplitude
25.06 7.63 13.67
24.21b 17.50b 15.77
24.39 13.97 11.69
24.76 4.77 10.27
22.73a,b 22.98b 17.68b
23.39a,b 36.09a,b,c 19.77a,b
Significant differences compared between 0 lux and 10 lux conditions (a), compared with w1118 (b) and compared between Elav/þ>hTauWT/ þ and Elav/þ>hTauRW/ þ (c) (p < 0.05).
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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Fig. 3. Altered sleep-wake behavior after exposure to dLAN in hTau flies. Sleep-wake behavior of hTau over-expressing flies under 0 lux (A) and 10 lux (B) with changes in total sleep (C), day light (D), night sleep (E) and sleep episodes at night (F) along with sleep deprivation (G) and sleep recovery (H). (*p < 0.05, **p < 0.01, ***p < 0.001).
3.4. Increased neurodegeneration in hTau-overexpressing flies after dim light exposure We observed significant increase in the pTau levels in the hTauRW-overexpressing flies after they were exposed to dLAN (Fig. 4A and B). To evaluate the effects of dim light exposure at night on neurodegeneration in the AD flies, the number of vacuoles was examined with brain histology [18]. Compared to hTauWT-overexpressing flies under 0 lux, significantly more vacuoles were found in the brains of the hTauRW-overexpressing flies (F(3,16) ¼ 14.33, p < 0.0001; Fig. 4C and D). After exposure to 10-lux dim light for 3 days, both hTauWT and hTauRW-overexpressing flies exhibited greater numbers of vacuoles compared with the 0-lux controls (Fig. 4C and D). Thus, these data suggested that dim light-induced
CR and sleep disruptions promote the accumulation of pTau and neurodegeneration in the brains of tauopathy/AD model flies. 4. Discussion We investigated the effects of dLAN on the rhythmic behaviors and disease symptoms of wild-type and AD model flies. Short-term exposure to a constant dim light of 10 lux altered the CR and sleepwake cycle of these flies. Tauopathy/AD flies exposed to dLAN showed more disruptions in their CRs and sleep-wake cycles and greater accumulation of pTau and traces of neurodegeneration in the brain compared with wild-type animals. Thus, these observations suggested that even light that is as dim as 10 lux at night was sufficient to influence rhythmic behaviors and aggravate disease
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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Fig. 4. Increased neurodegeneration and hyperphosphorylation of Tau in hTau-overexpressing flies after dim light exposure. (A and B) Increased pTau levels were observed in hTauRW-overexpressing flies under 10 lux conditions (*p < 0.05). (C) After exposure to 10 lux, the neuronal overexpression of hTau gave rise to increased number of vacuoles in the brain. The arrowheads indicate the vacuoles. (D) The quantification of vacuole number revealed that dim light exposure at night significantly increased the vacuoles in hTau-overexpressing flies (***p < 0.001).
symptoms in AD model animals. How does dim light disturb CRs and sleep? Fruit flies are very sensitive to light: light pulses of 0.02 lux (3 nW/cm2) cause phase advances in the CRs of wild-type flies [21]. Light response is mediated by the cryptochrome protein and light exposure reduces the expression of CRY [22]. Drosophila CRs are controlled by 6 groups of clock neurons, including the small and large ventral lateral neurons (s-LNv and l-LNv), dorsal lateral neurons (LNd), and dorsal neurons (DNs). Light is detected by the DN and LNd neurons expressing CRY, and the signal is transmitted to LNv neurons that secrete both CRY and the pigment dispersing factor (PDF) which controls morning peak [23]. Thus, light signals sensed by DN and LNd neurons in the morning are conveyed to LNv neurons to release PDF and increase locomotor activity. In the results of this study, night activity was enhanced whereas the morning peak was blunted by exposure to dLAN (Fig. 2B). Through the pathway connecting DN and LN neurons, dLAN might enhance fly activity at night and disturbs CRs and sleep patterns. In addition, the disrupted sleep behaviors found in the hTau-overexpressing flies after exposure to dim light might have been due to differential regulation of the PDF signaling.
Wittmann et al. [18] created the tauopathy/AD model flies by overexpressing the hTau gene in the Drosophila central nervous system. They observed enhanced neurodegeneration but not increased NFTs. When hTau was overexpressed with shaggy, the homologue of human GSK-3b in the brain, lifespan was reduced, and the number of vacuoles in the brain was increased. In addition, NFT-like structures (abnormal filaments) were observed in their brains [24]. As aging progresses, the phosphorylation of the serine 262 residue in the tau protein and the vacuolization of the brain tissue were enhanced in the tauopathy/AD flies [25]. Neurodegeneration resulting from the ectopic expression of hTau is related to cell death mechanisms, and significantly more cleaved caspase-3 is formed in the tauopathy flies [25]. More recently, Means et al. (2015) [26] have reported that the inhibition of Doubletime (Dbt) by spaghetti (CG13570) increases the levels of the activated initiator caspase Dronc, which cleaves Tau. We observed significant increases in pTau in the brains of the tauopathy flies (Figs. 1 and 4). Although it is uncertain whether this is related to the accumulation of hyperphosphorylated Tau or the formation of NFTs, the increase in the vacuole numbers in the tauopathy flies exposed to dLAN was probably due to the toxicity of the
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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accumulated pTau and/or the activation of the cell death pathway. However, activation of the TOR pathway seems to block neurodegeneration. During sleep deprivation or the awake state, phosphorylated AMPK inhibits TOR activity. Thus, it can be hypothesized that dLAN inhibits Dbt and/or activates AMPK to inhibit the TOR pathway and enhance neurodegeneration [27]. CRs and sleep behavior are influenced by aging and neurodegenerative diseases [28]. Inversely, the increased neurodegeneration in patients with AD affects CRs and sleep-wake behavior [29]. Patients with AD have increased wakefulness during the night [7] and therefore have a higher chance of being exposed to light at night. Because sleep plays an important role in clearing toxic materials that are accumulated during the daytime, any disturbances in sleep will eventually lead to the accumulation of these toxic materials [5], which in turn worsens the AD symptoms. In the current study, the hyperactivity of locomotion that was observed in the hTau-overexpressing flies under the LD condition was probably due to pTau production (Fig. 2B). The short-term exposure to continuous dLAN further increased the levels of pTau, which resulted in an increase in the severity of the disruptions in the CRs and sleep-wake behaviors (Fig. 4A and B). The sleep deprivation and rebound significantly changed in AD flies exposed to dim light at night (Fig. 3GeH), which suggested that pTau expression affected the sleep machinery. Thus, dLAN may result in a vicious cycle of pTau accumulation and poor sleep quality, which exacerbates neurodegeneration in AD model flies. The results of this study suggest that even 10 lux of dLAN would affect CR and sleep patterns in healthy individuals and more importantly aggravate the symptoms of patients suffering from neurodegenerative diseases, such as AD. As previously mentioned, light intensities over 10 lux in residential areas are defined as light pollution in many developed countries. Many workers in the shiftwork system are constantly exposed to light at night [10], which has an adverse effect on their CRs and metabolic disorders [12,21]. However, more studies are required to understand the effects of light that is dimmer than 10 lux at night. Meanwhile, many studies have been conducted to use light to improve the symptoms of dementia patients who have sleep disturbances [28,29], but the underlying mechanisms remain unclear. Author contributions Mari Kim and Manivannan Subramanian: acquisition of the data, analysis and interpretation of the data, preparation of the manuscript. Yun-Ho Cho and Gye-Hyeong Kim: acquisition of the data, analysis and interpretation of the data. Eunil Lee: concept of the study. Joong-Jean Park: concept and design of the study, preparation of the manuscript. Conflicts of interest All authors declare no conflict of interest. Acknowledgement This study was supported by grants from the National Research Foundation of Korea (NRF-2012M3A9B6055351 and NRF2017R1D1A1B03033648 to J-J.P; NRF-2016R1D1A102937060 to M.K. and E.L.) and the Korean Environmental Industry and Technology Institute (KEITI) through environmental health action program (EHAP) (RE201704026 to M.K.) funded by Korea Ministry of Environment.
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Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease Mari Kim b, 1, Manivannan Subramanian a, 1, Yun-Ho Cho a, Gye-Hyeong Kim a, Eunil Lee b, Joong-Jean Park a, * a b
Department of Physiology, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea Department of Preventive Medicine, College of Medicine, Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2017 Accepted 4 December 2017 Available online xxx
The accumulation and aggregation of phosphorylated tau proteins in the brain are the hallmarks for the onset of Alzheimer's disease (AD). In addition, disruptions in circadian rhythms (CRs) with altered sleepwake cycles, dysregulation of locomotion, and increased memory defects have been reported in patients with AD. Drosophila flies that have an overexpression of human tau protein in neurons exhibit most of the symptoms of human patients with AD, including locomotion defects and neurodegeneration. Using the fly model for tauopathy/AD, we investigated the effects of an exposure to dim light at night on AD symptoms. We used a light intensity of 10 lux, which is considered the lower limit of light pollution in many countries. After the tauopathy flies were exposed to the dim light at night for 3 days, the flies showed disrupted CRs, altered sleep-wake cycles due to increased pTau proteins and neurodegeneration, in the brains of the AD flies. The results indicate that the nighttime exposure of tauopathy/AD model Drosophila flies to dim light disrupted CR and sleep-wake behavior and promoted neurodegeneration. © 2017 Elsevier Inc. All rights reserved.
Keywords: Alzheimer's disease (AD) Circadian rhythm (CR) Sleep Dim light at night (dLAN) Neurodegeneration Drosophila melanogaster
1. Introduction Alzheimer's disease (AD) is one of the most common forms of dementia all over the world. The onset of AD is associated with the accumulation of b-amyloid peptide (Ab) and/or phosphorylated tau [1]. Tau stabilizes microtubules, which are involved in neuronal axon transport [2]. In patients with AD, hyperphosphorylated tau is segregated from microtubules, which results in the loss of microtubule integrity. In addition, hyperphosphorylated tau accumulates in patients with AD and forms intracellular neurofibrillary tangles (NFTs), which become neurotoxic and eventually cause neurodegeneration [3]. Rhythmic behaviors are important in normal physiological processes. Two common and interacting rhythmic behaviors are circadian rhythms (CRs) and sleep-wake behaviors. CRs are involved in the regulation of the sleep-wake behavior, and disruptions in CRs eventually result in changes in sleep. Normal sleep
* Corresponding author. E-mail address: [email protected] (J.-J. Park). 1 Contributed equally on this work.
is required to restore neural plasticity and function [4] and clear toxic compounds that accumulate during the daytime [5]. Studies conducted using AD model mice have shown that the CRs of the mice are altered by the accumulation of Ab and the formation of NFTs [6]. Sleep is profoundly affected in patients with AD showing altered sleep-wake behavior with reduced night sleep. Mouse models exhibiting AD phenotypes also show decreased sleep at night [6]. Thus, sleep disturbances are considered an indicator of the pathogenesis of AD [7]. Even in healthy individuals, decreased sleep enhances the accumulation of Ab peptides and the formation of NFTs [8]. Thus, sleep and AD are interlinked, and any perturbations of one will affect the other [9]. The interference of CRs and/or sleep by light at night is referred to as light pollution, which has recently emerged as a major health problem in many developed countries, where any nighttime exposure to light that is over 10 lux in a residential area is prohibited. In modern industrialized societies, shift workers are continuously exposed to light at night, which disrupts their CRs and sleep-wake behaviors [10]. Shift workers have recently been reported to be more susceptible to cardiovascular disease, diabetes, obesity, cancer, and metabolic disorders [11,12]. Studies conducted
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Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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on mice have also shown that disruptions of CRs by exposure to dim light (10 lux) at night leads to increased tumorigenesis with reduced lifespans [11]. Because CRs are regulated by the nervous system, any negative effects of CR disruptions on other systems should appear rapidly. Drosophila melanogaster has been serving as a useful model system for investigating the genetic bases of CRs and sleep [13] and the pathogenesis of AD [14,15]. Previous studies on Drosophila have reported that exposure to moonlight at night alters CRs [16]. However, it remains unclear whether exposure to 10 lux of dLAN affects rhythmic behaviors and/or neurodegeneration in AD model organisms. In this study, we investigated the effects of short-term exposures to dim light on physiology in AD flies and found that AD flies were more vulnerable to the negative effects of the dim light exposure.
used as a loading control. 2.5. Immunohistochemistry Adult fly heads were fixed in 4% paraformaldehyde and washed with PBS. The brains were blocked with 5% normal goat serum in PBS containing 0.1% Triton-X 100 (PBST) and incubated with murine anti-tau and rabbit anti-pTau antibodies for 48 h at 4 C. After washing with PBST, the samples were incubated with an antimouse secondary antibody conjugated with Alexa Fluor 488 and an anti-rabbit secondary antibody conjugated with Alexa Fluor 524 overnight at 4 C. After washing, brains were mounted using Vectashield (Thermo Fisher Scientific Inc.) and images were acquired using confocal microscopy (Carl Zeiss Microscopy). 2.6. Brain histology
2. Materials and methods 2.1. Drosophila strains The flies were cultured on cornmeal-based standard food under a 12:12-h light-dark (LD) cycle at 25 C with 40e60% relative humidity [17]. Elav-GAL4 (ElavC155-GAL4) and GMR-GAL4 fly stocks were obtained from Bloomington Drosophila Stock Center (Bloomington, IN, USA). Other stocks used were UAS-hTauWT, UAS-hTauRW [18], and w1118 (þ/þ) for the wild type. 2.2. Circadian rhythms and sleep Freshly eclosed Drosophila males were collected and transferred into 3-mm-diameter glass tubes containing food at one end. These flies were monitored for 6 days using the Drosophila Activity Monitoring System (TriKinetics, Inc., Waltham, MA, USA) in an incubator with a 12:12-h LD cycle. For the first 3 days, the flies were maintained under a normal LD cycle, and then exposed 10 lux during the dark cycle for the next 3 days. Locomotor activity was recorded for 30-min intervals, and rhythmicity and free-running periods were calculated using a cosinor analysis. Sleep behaviors were analyzed during the 3rd day of each lighting pattern (LD3 or LL3) using pySolo software. Sleep deprivation was calculated by subtracting the sleep time recorded on the third day (LD3 and LL3) during the 10-lux light exposure from the sleep time recorded during 0 lux in each treatment group. Similarly, the amount of sleep recovered from ZT09 to ZT21 the day after the third day was calculated [19].
The Drosophila heads that were fixed in 4% paraformaldehyde were embedded in paraffin blocks and sectioned at a thickness of 6 mm. These sections were mounted on slides and stained using hematoxylin and eosin. Quantification of the neurodegeneration was performed as mentioned by Lijima et. Al (2008) [14]. 2.7. Climbing assay Around 20 flies of each genotype were placed in a 50-mL glass mass cylinder and gently tapped to the bottom. The number of flies that climbed to the top was counted after 10 s. The data were acquired from three independent sets and then analyzed and plotted. 2.8. Data analysis The longevity data were used to generate Kaplan-Meier survival plots, and median lifespan was calculated. Statistical significance between the genotypes was analyzed using Log-Rank tests and the Prism software. For the data on the CR, sleep parameters, sleep deprivation and sleep recovery were analyzed using a 2-way analysis of variance with the main factors of genotype and light condition. The number of vacuoles and pTau intensity of the western blots were analyzed using a 2-way repeated analysis of variance. P values less than 0.05 were considered significant, and the data are presented as mean ± standard error of the mean. 3. Results 3.1. Accumulation of hTau negatively affected lifespan
2.3. Lifespan assay The flies were raised at 25 C under 50% relative humidity and a 12:12-h LD cycle, and the food vials were replaced every 2e3 days [17]. Dead flies were counted every 2e3 days, and the survival curves were plotted using Prism software (v6.05; GraphPad Software). 2.4. Western blotting Adult fly heads were homogenized in HEPES-EDTA lysis buffer and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and loaded onto SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk and incubated overnight at 4 C with anti-tau and antipTau antibodies (both 1:1000; Thermo Fisher Scientific) followed by incubation with secondary antibody (1:10,000; Cell Signaling Technology) and visualized with chemiluminescent detection. A mouse b-actin antibody (1:1000; Cell Signaling Technology) was
Studies of hTau have shown that the accumulation of hTau in fly brains shortens their lifespan and increases neurodegeneration in Drosophila [18]. Two types of human tau (hTau) protein were overexpressed: wild-type hTau (hTauWT) and mutant hTau, in which Arg406 was replaced by Trp (hTauRW). Accumulation of hTau proteins in the brain were confirmed with both immunohistochemistry (Fig. 1A) and western blotting (Fig. 1B). Flies overexpressing hTauWT exhibited the distinct rough eye phenotype under control of either the Elav-GAL4 or the GMR-GAL4 (Fig. 1C). The rough eye phenotype was exacerbated in hTauRW flies overexpressed both in neurons and the eye. Flies overexpressing hTauWT or hTauRW, in neurons showed significant shorter lifespans compared to controls (p < 0.001, Fig. 1D). The median lifespan (20 days) of the hTauRW-overexpressing flies was significantly shorter than that (39 days) of the hTauWT-overexpressing flies (p < 0.001, Fig. 1D). Furthermore, these results suggested that the neuronal overexpression of the hTau gene enhanced the formation of pTau and reduced lifespan in Drosophila.
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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Fig. 1. Overexpression of hTau results in the accumulation of pTau and reduces lifespan. (A) Visualization of hTau in brains by immunohistochemistry. (B) Detection of hTau and pTau by western blotting. (C) Rough eye phenotypes in flies expressing hTauWT and hTauRW using GMR-GAL4 and Elav-GAL4. (D) The overexpression of either hTauWT or hTauRW in the brain using the pan-neuronal Elav-GAL4 (Elav/þ>hTauWT/þ; Elav/þ>hTauRW/þ) significantly reduced lifespan, compared to controls.
3.2. Negative effects of exposure to dim light at night on circadian rhythm in hTau-overexpressing flies Recent studies of AD models have investigated the disruptions in circadian behavior and locomotion in AD [20]. Circadian oscillations are controlled by both external and internal factors. To investigate whether internal rhythmicity was maintained in tauopathy flies, hTau-overexpressing flies were conditioned to a darkdark (DD) cycle, and their CRs were analyzed. Under the DD condition, the flies overexpressing hTauWT or hTauRW in neurons maintained their CRs, which were similar to those of the wild-type flies (F(2,54) ¼ 2.182, p ¼ 0.1227; Fig. 2A). Circadian rhythmicity was calculated using cosinor analyses, which measures period (time taken to complete one cycle in 24 h), amplitude (maximum activity in 24 h), and robustness (persistent behavior of locomotion). The cosinor analyses of their CRs showed that the period and amplitude of the locomotion behavior did not differ significantly between the LD and DD conditions in the hTau-overexpressing flies and the robustness under the LD condition was similar to that of the DD condition (Table 1). These results suggested that the intrinsic circadian clock mechanism was intact and present in both the hTauWT and hTauRW-overexpressing flies. Because the internal clocks functioned normally under the DD condition in the AD model flies, these flies were subjected to variations in external cues by exposing them to dLAN (10 lux). During exposure to 0 lux, normal behavior with the characteristic morning and evening peaks according to the lighting schedule was observed in the wild-type flies (Fig. 2B). However, the morning peak was reduced due to increased nocturnal locomotor activity on exposure to dLAN (Fig. 2B), whereas the CR period was unaffected (Table 2). Interestingly, under 10 lux, period was significantly decreased in the hTauWT-overexpressing flies (F(2,52) ¼ 7.672, p ¼ 0.0012; Table 2), whereas their robustness and amplitude were not significantly different (Table 2). In hTauRW-overexpressing flies, the dim light exposure resulted in a significant reduction in period (F(1,52) ¼ 9.022, p ¼ 0.0041; Table 2) and increased robustness
(F(2,52) ¼ 12.52, p < 0.0001; Table 2). Under dLAN, flies overexpressing hTauRW showed increased circadian disruption (Fig. 2B) with increased robustness. Despite these changes, climbing defects were observed only in hTau-overexpressing flies, and dim light at night did not affect their antigeotactic locomotor ability (Fig. 2C), which suggested that dim light at night only affected the circadian locomotor activity. 3.3. Disruption of sleep-wake behavior after exposure to dLAN in hTau-overexpressing flies Sleep, which is regulated by both circadian and homeostatic mechanisms, is disrupted in patients with AD. Sleep-wake behaviors were further assessed in hTau-overexpressing flies. Under the 0-lux condition, the wild-type flies exhibited normal sleep-wake behavior (Fig. 3A). However, they showed altered sleep-wake behaviors with decreased night-time sleep after exposure to dLAN (Fig. 3B) compared to flies under LD condition (F(5,45) ¼ 5.957, p ¼ 0.0003; Fig. 3E). However, they showed increased compensatory sleep during the daytime due to their sleep disturbances induced by the dLAN (F(5,45) ¼ 12.39, p < 0.0001; Fig. 3D). Flies overexpressing hTauWT showed no changes in sleep-wake behavior after exposure to dLAN (Fig. 3CeF). However, flies overexpressing hTauRW exhibited altered sleep-wake behavior (Fig. 3A) with significant reduction in total sleep (F(5,45) ¼ 9.536, p < 0.00019; Fig. 3C), night sleep (F(5,45) ¼ 5.937, p ¼ 0.0003; Fig. 3E), and sleep episodes at night (F(5,45) ¼ 2.736, p ¼ 0.0305; Fig. 3F) on exposure to dLAN. When the wild-type flies were exposed to the dLAN, their sleep was significantly deprived, but it was recovered the next day (Fig. 3G and H). However, the flies overexpressing hTauWT had significantly reduced sleep deprivation (p ¼ 0.0065; Fig. 3G) and recovery (p ¼ 0.013; Fig. 3H). For the hTauRW-overexpressing flies, considerable sleep deprivation was found, and the sleep amount that was recovered the next day was significantly reduced (p ¼ 0002; Fig. 3H). These data suggested that the exposure of AD flies to dim light affected their CR and sleep-wake behaviors.
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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Fig. 2. Disruption of CR in flies overexpressing hTau in the brain after exposure to dLAN. (A) CR in w1118 flies and hTau overexpressing flies under LD and DD conditions with cosinor analysis (Table 1). (B) CR in w1118 flies and hTau overexpressing flies under 0 lux and 10 lux conditions with cosinor analysis (Table 2). (C) Reduction of anti-geotactic locomotor (climbing) activity in hTau overexpressing flies under both 0 lux and 10 lux.
Table 1 Circadian rhythmicity under constant darkness condition. Light condition
LD
Genotype
w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
DD w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
Period Robustness Amplitude
25.40 1.53 11.00
24.20 3.72b 11.20
24.20 8.79b 16.08
22.4a 2.75 9.75
23.60 4.09 9.07
24.79b 16.18b,c 15.71
Significant differences compared between LD and DD conditions (a), compared with w1118 (b) and compared between Elav/þ>hTauWT/ þ and Elav/þ>hTauRW/ þ (c) (p < 0.05).
Table 2 Circadian rhythmicity on exposure to dim (10 lux) light at night. Light condition
0 lux
Genotype
w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
10 lux w1118 (þ/þ)
Elav/þ > hTauWT/þ
Elav/þ > hTauRW/þ
Period Robustness Amplitude
25.06 7.63 13.67
24.21b 17.50b 15.77
24.39 13.97 11.69
24.76 4.77 10.27
22.73a,b 22.98b 17.68b
23.39a,b 36.09a,b,c 19.77a,b
Significant differences compared between 0 lux and 10 lux conditions (a), compared with w1118 (b) and compared between Elav/þ>hTauWT/ þ and Elav/þ>hTauRW/ þ (c) (p < 0.05).
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Fig. 3. Altered sleep-wake behavior after exposure to dLAN in hTau flies. Sleep-wake behavior of hTau over-expressing flies under 0 lux (A) and 10 lux (B) with changes in total sleep (C), day light (D), night sleep (E) and sleep episodes at night (F) along with sleep deprivation (G) and sleep recovery (H). (*p < 0.05, **p < 0.01, ***p < 0.001).
3.4. Increased neurodegeneration in hTau-overexpressing flies after dim light exposure We observed significant increase in the pTau levels in the hTauRW-overexpressing flies after they were exposed to dLAN (Fig. 4A and B). To evaluate the effects of dim light exposure at night on neurodegeneration in the AD flies, the number of vacuoles was examined with brain histology [18]. Compared to hTauWT-overexpressing flies under 0 lux, significantly more vacuoles were found in the brains of the hTauRW-overexpressing flies (F(3,16) ¼ 14.33, p < 0.0001; Fig. 4C and D). After exposure to 10-lux dim light for 3 days, both hTauWT and hTauRW-overexpressing flies exhibited greater numbers of vacuoles compared with the 0-lux controls (Fig. 4C and D). Thus, these data suggested that dim light-induced
CR and sleep disruptions promote the accumulation of pTau and neurodegeneration in the brains of tauopathy/AD model flies. 4. Discussion We investigated the effects of dLAN on the rhythmic behaviors and disease symptoms of wild-type and AD model flies. Short-term exposure to a constant dim light of 10 lux altered the CR and sleepwake cycle of these flies. Tauopathy/AD flies exposed to dLAN showed more disruptions in their CRs and sleep-wake cycles and greater accumulation of pTau and traces of neurodegeneration in the brain compared with wild-type animals. Thus, these observations suggested that even light that is as dim as 10 lux at night was sufficient to influence rhythmic behaviors and aggravate disease
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Fig. 4. Increased neurodegeneration and hyperphosphorylation of Tau in hTau-overexpressing flies after dim light exposure. (A and B) Increased pTau levels were observed in hTauRW-overexpressing flies under 10 lux conditions (*p < 0.05). (C) After exposure to 10 lux, the neuronal overexpression of hTau gave rise to increased number of vacuoles in the brain. The arrowheads indicate the vacuoles. (D) The quantification of vacuole number revealed that dim light exposure at night significantly increased the vacuoles in hTau-overexpressing flies (***p < 0.001).
symptoms in AD model animals. How does dim light disturb CRs and sleep? Fruit flies are very sensitive to light: light pulses of 0.02 lux (3 nW/cm2) cause phase advances in the CRs of wild-type flies [21]. Light response is mediated by the cryptochrome protein and light exposure reduces the expression of CRY [22]. Drosophila CRs are controlled by 6 groups of clock neurons, including the small and large ventral lateral neurons (s-LNv and l-LNv), dorsal lateral neurons (LNd), and dorsal neurons (DNs). Light is detected by the DN and LNd neurons expressing CRY, and the signal is transmitted to LNv neurons that secrete both CRY and the pigment dispersing factor (PDF) which controls morning peak [23]. Thus, light signals sensed by DN and LNd neurons in the morning are conveyed to LNv neurons to release PDF and increase locomotor activity. In the results of this study, night activity was enhanced whereas the morning peak was blunted by exposure to dLAN (Fig. 2B). Through the pathway connecting DN and LN neurons, dLAN might enhance fly activity at night and disturbs CRs and sleep patterns. In addition, the disrupted sleep behaviors found in the hTau-overexpressing flies after exposure to dim light might have been due to differential regulation of the PDF signaling.
Wittmann et al. [18] created the tauopathy/AD model flies by overexpressing the hTau gene in the Drosophila central nervous system. They observed enhanced neurodegeneration but not increased NFTs. When hTau was overexpressed with shaggy, the homologue of human GSK-3b in the brain, lifespan was reduced, and the number of vacuoles in the brain was increased. In addition, NFT-like structures (abnormal filaments) were observed in their brains [24]. As aging progresses, the phosphorylation of the serine 262 residue in the tau protein and the vacuolization of the brain tissue were enhanced in the tauopathy/AD flies [25]. Neurodegeneration resulting from the ectopic expression of hTau is related to cell death mechanisms, and significantly more cleaved caspase-3 is formed in the tauopathy flies [25]. More recently, Means et al. (2015) [26] have reported that the inhibition of Doubletime (Dbt) by spaghetti (CG13570) increases the levels of the activated initiator caspase Dronc, which cleaves Tau. We observed significant increases in pTau in the brains of the tauopathy flies (Figs. 1 and 4). Although it is uncertain whether this is related to the accumulation of hyperphosphorylated Tau or the formation of NFTs, the increase in the vacuole numbers in the tauopathy flies exposed to dLAN was probably due to the toxicity of the
Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021
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accumulated pTau and/or the activation of the cell death pathway. However, activation of the TOR pathway seems to block neurodegeneration. During sleep deprivation or the awake state, phosphorylated AMPK inhibits TOR activity. Thus, it can be hypothesized that dLAN inhibits Dbt and/or activates AMPK to inhibit the TOR pathway and enhance neurodegeneration [27]. CRs and sleep behavior are influenced by aging and neurodegenerative diseases [28]. Inversely, the increased neurodegeneration in patients with AD affects CRs and sleep-wake behavior [29]. Patients with AD have increased wakefulness during the night [7] and therefore have a higher chance of being exposed to light at night. Because sleep plays an important role in clearing toxic materials that are accumulated during the daytime, any disturbances in sleep will eventually lead to the accumulation of these toxic materials [5], which in turn worsens the AD symptoms. In the current study, the hyperactivity of locomotion that was observed in the hTau-overexpressing flies under the LD condition was probably due to pTau production (Fig. 2B). The short-term exposure to continuous dLAN further increased the levels of pTau, which resulted in an increase in the severity of the disruptions in the CRs and sleep-wake behaviors (Fig. 4A and B). The sleep deprivation and rebound significantly changed in AD flies exposed to dim light at night (Fig. 3GeH), which suggested that pTau expression affected the sleep machinery. Thus, dLAN may result in a vicious cycle of pTau accumulation and poor sleep quality, which exacerbates neurodegeneration in AD model flies. The results of this study suggest that even 10 lux of dLAN would affect CR and sleep patterns in healthy individuals and more importantly aggravate the symptoms of patients suffering from neurodegenerative diseases, such as AD. As previously mentioned, light intensities over 10 lux in residential areas are defined as light pollution in many developed countries. Many workers in the shiftwork system are constantly exposed to light at night [10], which has an adverse effect on their CRs and metabolic disorders [12,21]. However, more studies are required to understand the effects of light that is dimmer than 10 lux at night. Meanwhile, many studies have been conducted to use light to improve the symptoms of dementia patients who have sleep disturbances [28,29], but the underlying mechanisms remain unclear. Author contributions Mari Kim and Manivannan Subramanian: acquisition of the data, analysis and interpretation of the data, preparation of the manuscript. Yun-Ho Cho and Gye-Hyeong Kim: acquisition of the data, analysis and interpretation of the data. Eunil Lee: concept of the study. Joong-Jean Park: concept and design of the study, preparation of the manuscript. Conflicts of interest All authors declare no conflict of interest. Acknowledgement This study was supported by grants from the National Research Foundation of Korea (NRF-2012M3A9B6055351 and NRF2017R1D1A1B03033648 to J-J.P; NRF-2016R1D1A102937060 to M.K. and E.L.) and the Korean Environmental Industry and Technology Institute (KEITI) through environmental health action program (EHAP) (RE201704026 to M.K.) funded by Korea Ministry of Environment.
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Please cite this article in press as: M. Kim, et al., Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.12.021